DNA immunization with recombinase/transposase

Abstract

The present invention relates to improved methods to immunize/vaccinate or stimulate the immune system of animals, including humans, using vectors containing expression cassettes that encode for the DNA of one or more protein/peptide antigens and/or adjuvants, in particular, cytokines like GMCSF, Flt3L, interleukins, and the like, which can be encoded by DNA as well, also recombinase mediated integration. Adjuvants known to increase immune responses following DNA vaccination. In addition, the vectors contain one or more sites recognized by a recombinase/transposase, which catalyzes the insertion of the vector into the genome of transfected cells. Stable integration of the plasmid vector into the genome of transfected cells results in higher and longer-lasting expression of the encoded protein(s), and increases the immune response in the vaccinated animal. The present invention also relates to adjuvant compositions comprising the novel polypeptide, rabbit GMCSF, for boosting antibody production in rabbits.

1 . A rabbit GMCSF polypeptide of SEQ ID NO: 1.  
     
     
         2 . A chimeric molecule comprising the polypeptide of  claim 1  fused to a heterologous amino acid sequence.  
     
     
         3 . The chimeric molecule of  claim 2  wherein said heterologous amino acid sequence is an epitope sequence.  
     
     
         4 . The chimeric molecule of  claim 2  wherein said heterologous amino acid sequence is an immunoglobulin sequence.  
     
     
         5 . The chimeric molecule of  claim 4  wherein said immunoglobulin sequence is an Fc region of an immunoglobulin.  
     
     
         6 . A nucleotide sequence comprising a nucleotide sequence encoding the rabbit GMCSF polypeptide of  claim 1 .  
     
     
         7 . A vector or expression cassette comprising the nucleotide sequence of  claim 6 .  
     
     
         8 . An isolated host cell transformed with the nucleic acid of  claim 6 .  
     
     
         9 . A method for immunizing or vaccinating an animal, said method comprising: 
 (i) administering at least one DNA construct comprising at least one expression cassette that encodes for an antigen, and a first recombination site;    (ii) stimulating the immune system of said animal by administering at least one adjuvant to said animal; and,    (iii) administering a recombinase that mediates the integration of said expression cassette into the genome of said animal comprising a second recombination site,    wherein, said antigen is expressed.    
     
     
         10 . The method of  claim 9  wherein said adjuvant is introduced as a polypeptide.  
     
     
         11 . The method of  claim 9  wherein the administration of the adjuvant comprises introducing to said animal: (i) said adjuvant as a DNA construct comprising an expression cassette that encodes for said adjuvant, and, a first recombination site; and, (ii) a recombinase that mediates the integration of said expression cassette into the genome of said animal comprising a second recombination site, wherein, said adjuvant is expressed.  
     
     
         12 . The method of  claim 9  wherein said antigen(s) is/are selected from the group consisting of viral, bacterial, fungal, protozoal antigens and antigens associated with diseases like infection, inflammation, cancer, asthma/allergy, autoimmune diseases, multiple sclerosis, sepsis/toxic shock, rheumatoid arthritis, allograft rejections, psoriasis, etc.  
     
     
         13 . The method of  claim 9  wherein said antigen is CD20.  
     
     
         14 . The method of  claim 11  wherein said adjuvant(s) is/are selected from the group consisting of GMCSF, Flt3L, interleukins like L-1α and β, IL-2, IL-12, IL-15, IL-18, IL-4, IL-5, IL-6, IL-10, TNF-α, TNF-β, IFN-γ, etc. and co-stimulatory molecules like TCA3, CD80 (B7.1), CD86 (B7.2), CD40 ligand (CD154), MCP-1, MIP-1α, β, RANTES, etc.  
     
     
         15 . The method of  claim 11  wherein said adjuvant is the rabbit GMCSF of SEQ ID NO:1.  
     
     
         16 . The method of  claim 11  wherein said antigen-encoding expression cassette and said adjuvant-encoding expression cassette are part of one DNA construct.  
     
     
         17 . The method of  claim 11  wherein said antigen-encoding expression cassette and said adjuvant-encoding expression cassette are on separate DNA constructs.  
     
     
         18 . The method of  claim 9  or  11  wherein said recombinase is administered as a polypeptide.  
     
     
         19 . The method of  claim 9  or  11  wherein said recombinase is administered as a messenger RNA molecule encoding said recombinase.  
     
     
         20 . The method of  claim 9  or  11  wherein said recombinase is administered as a DNA construct comprising an expression cassette encoding said recombinase.  
     
     
         21 . The method of  claim 9  or  11  wherein said recombinase is a site-specific recombinase expressed by a phage.  
     
     
         22 . The method of  claim 21  wherein said recombinase is selected from the group consisting of φC31, phage R4 and TP901-1.  
     
     
         23 . The method of  claim 21  wherein said site specific recombinase is selected from the group consisting of a Cre-recombinase, a Cre-like recombinase, a Flp recombinase and an R recombinase.  
     
     
         24 . The method of  claim 9  or  11  wherein said recombinase is a transposase or a retrotransposase.  
     
     
         25 . The method of  claim 24 , wherein said transposase is selected from the group consisting of AC7, Tn5, Tn916, Tn951, Tn1721, Tn2410, Tn1681, Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn9, Tn10, Tn30, Tn101, Tn903, Tn501, Tn1000, Tn1681, tn2901, AC transposons, Mp transposons, Spm transposons, En transposons, Dotted transposons, Mu transposons, Ds transposons, En transposons, and I transposons.  
     
     
         26 . The method of  claim 24 , wherein said transposase is a eukaryotic transposase.  
     
     
         27 . The method of  claim 9  or  11  wherein said first and second recombination sites share at least 90% sequence identity.  
     
     
         28 . The method of  claim 9  or  11  wherein said first and second recombination sites share less than 90% sequence identity.  
     
     
         29 . The method of  claim 9  or  11  wherein said first recombination site comprises a bacterial genomic recombination site and said second recombination site comprises a phage recombination site.  
     
     
         30 . The method of  claim 29  wherein said bacterial genomic recombination site is attB and said phage recombination site is attP.  
     
     
         31 . The method of  claim 29 , wherein said first recombination site comprises an attB site, and said second recombination site comprises a pseudo-attP site.  
     
     
         32 . The method of  claim 29 , wherein said first recombination site comprises a pseudo-attB site, and said second recombination site comprises an attP site.  
     
     
         33 . The method of  claim 9  or  11 , wherein said recombinase-mediated recombination results in a site that is no longer a substrate for the recombinase.  
     
     
         34 . The method of  claim 9  or  11  wherein said DNA construct(s) is/are circular.  
     
     
         35 . The method of  claim 9  or  11  wherein said DNA construct(s) is/are linear.  
     
     
         36 . The method of  claim 9  wherein said animal is non-human mammal.  
     
     
         37 . The method of  claim 36  wherein said non-human mammal is a rabbit.  
     
     
         38 . The method of  claim 9  wherein said animal is a human.  
     
     
         39 . A method for producing antibodies in an animal, comprising: 
 (i) administering at least one DNA expression cassette encoding an antigen, a recombination site, and a recombinase that mediates the integration of said DNA expression cassette into the genome of said animal;    (ii) optionally administering at least one adjuvant;    (iii) harvesting a serum sample after several days from said animal;    (iv) identifying and optionally, purifying antibodies to said administered antigen(s) from said serum sample.    
     
     
         40 . The method of  claim 39  wherein said adjuvant is introduced as a polypeptide.  
     
     
         41 . The method of  claim 39  wherein said adjuvant is introduced as a nucleic acid comprising an expression cassette encoding said adjuvant, a recombination site and a recombinase that mediates the integration of said adjuvant encoding-DNA expression cassette into the genome of said animal.  
     
     
         42 . The method of  claim 39  wherein said animal is a human.  
     
     
         43 . The method of  claim 39  wherein said animal is a non-human mammal.  
     
     
         44 . The method of  claim 39  wherein said animal is a non-human transgenic animal carrying an exogenous immunoglobulin translocus.  
     
     
         45 . The method of  claim 44  wherein said exogenous immunoglobulin translocus is a human or humanized immunoglobulin heavy and/or light chain sequence.  
     
     
         46 . The method of  claim 44  wherein said non-human transgenic animal is a gene converting animal.  
     
     
         47 . The method of  claim 44  wherein said non-human transgenic animal is selected from the group consisting of rodents, rabbits, birds including chickens, turkeys, ducks and geese.  
     
     
         48 . The method of  claim 43  wherein said non-human mammal is a rabbit.  
     
     
         49 . The method of  claim 39  wherein said antigen is selected from the group consisting of viral, bacterial, fungal, protozoal antigens and antigens associated with diseases like cancer, asthma/allergy, autoimmune diseases, multiple sclerosis, inflammation/sepsis/toxic shock, rheumatoid arthritis, allograft rejections, psoriasis, etc.  
     
     
         50 . The method of  claim 49  wherein said antigen is CD20.  
     
     
         51 . The method of  claim 39  wherein said adjuvant is selected from the group consisting of GMCSF, Flt3L, interleukins like IL-1α and β, IL-2, IL-12, IL-15, IL-18, IL-4, IL-5, IL-6, IL-10, TNF-α, TNF-β, IFN-γ, etc. and co-stimulatory molecules like TCA3, CD80 (B7.1), CD86 (B7.2), CD40 ligand (CD154), MCP-1, MIP-1α, β, RANTES, etc.  
     
     
         52 . The method of  claim 39  wherein said adjuvant is a rabbit GMCSF of SEQ ID NO: 1.